RESUMO
Allorecognition is known to involve a large number of lymphocytes carrying diverse T-cell receptor repertoire. Thus, one way to understand allorecognition and rejection mechanisms is via high-throughput sequencing of T-cell receptors. In this study, in order to explore and systematize the properties of the alloreactive T-cell receptor repertoire, we modeled direct and indirect allorecognition pathways using material from inbred mice in vitro and in vivo. Decoding of the obtained T-cell receptor genes using high-throughput sequencing revealed some features of the alloreactive repertoires. Thus, alloreactive T-cell receptor repertoires were characterized by specific V-gene usage patterns, changes in CDR3 loop length, and some amino acid occurrence probabilities in the CDR3 loop. Particularly pronounced changes were observed for directly alloreactive clonotypes. We also revealed a clustering of directly and indirectly alloreactive clonotypes by their ability to bind a single antigen; amino acid patterns of the CDR3 loop of alloreactive clonotypes; and the presence in alloreactive repertoires of clonotypes also associated with infectious, autoimmune, and tumor diseases. The obtained results were determined by the modeling of the simplified allorecognition reaction in inbred mice in which stimulation was performed with a single MHCII molecule. We suppose that the decomposition of the diverse alloreactive TCR repertoire observed in humans with transplants into such simple reactions will help to find alloreactive repertoire features; e.g., a dominant clonotype or V-gene usage pattern, which may be targeted to correct the entire rejection reaction in patients. In this work, we propose several technical ways for such decomposition analysis, including separate modeling of the indirect alloreaction pathway and clustering of alloreactive clonotypes according to their ability to bind a single antigen, among others.
RESUMO
BACKGROUND: B220+CD11c+plasmacytoid DCs(pDCs) are known to participate in the negative selection and central tolerance induction by the capturing of self-antigens in peripheral tissues and further migration to the thymus using the CCL25-CCR9 chemotaxis axis. AIM: Here we investigate the possibility of DCs migration stimulation to the thymus by the transfection with plasmid DNA-constructs encoding CCR9(pmaxCCR9) to develop a system for desired antigen delivery to the thymus for central tolerance induction. METHODS: Dendritic cells(DCs) cultures were generated from UBC-GFP mice bone marrow cells expressing green fluorescent protein using the rmFlt3-L. DCs cultures were transfected with pmaxCCR9 by electroporation. The efficiency of electroporation was confirmed by RT-qPCR and flow cytometry. The migration of electroporated DCs was assessed in vitro and in vivo. RESULTS: Dendritic cells(DCs) cultures obtained from UBC-GFP mice contained both B220+pDCs and SIRPa+cDC2. According to the RT-qPCR assay, the electroporation of obtained DCs cultures with pmaxCCR9 resulted in a 94.4-fold increase of RNA encoding CCR9 compared with non-electroporated cultures. Flow cytometry data showed that DCs cultures electroporated with pmaxCCR9 contained a significantly higher frequency of DCs carrying significantly higher levels of surface CCR9. Migration dynamics of obtained DCs analyzed in vitro showed that pmaxCCR9 electroporated DCs migrated significantly more active to CCL25 and thymic cells than non-electroporated and mock-electroporated DCs. In vivo, 30 days after injection, the relative amount of the DCs electroporated with pmaxCCR9 and pmaxMHC encoding antigenic determinants in the mice thymuses was 2.02-fold higher than the relative amount of the DCs electroporated with control plasmid. CONCLUSION: Thus, the electroporation of murine DCs with pmaxCCR9 stimulated its migration to CCL25 and thymic cells in vitro as well as to the thymus in vivo. The obtained DCs loaded with a desired antigen may be suggested for further evaluation of central tolerance induction ability in in vivo models of autoimmune diseases and transplantation.
